Pharmaceutical Compositions and Methods for Digesting Atherosclerotic Plaques

ABSTRACT

Disclosed are pharmaceutical compositions and methods for digesting atherosclerotic plaques in a patient in need thereof. The compositions include and the methods utilize a mixture of collagenases for digesting plaques and optionally may include or utilize additional agents such as cyclodextrins, chelating agents, and tissue plasminogen activator.

BACKGROUND

The field of the invention is related to cardiovascular diseaseincluding cardiovascular disease secondary to atheroschlerosis andcompositions and methods for treating or preventing cardiovasculardisease secondary to atherosclerosis. The compositions and methods maybe utilized for digesting atheroschlerotic plaques.

Cardiovascular disease secondary to atherosclerosis is the leading causeof death in the United States, accounting for 60% of total mortality in2002. (See Rosamond W, et al. Circulation 2007; 115(5):E69-E171.)Approximately 16 million people in the United States have clinicallymanifested coronary heart disease, approximately 8 million haveperipheral arterial disease, and over 5 million are stroke survivors.(See id.)

Atherosclerotic plaques evolve through a continuum of histologicalchanges. Inflammation starts when endothelial cells become activated andsecrete adhesion molecules, and the vascular smooth muscle cells (VSMC)secrete chemokines and chemoattractants. Together, these agents attractmonocytes, lymphocytes, mast cells, and neutrophils into the arterialwall. (See Katsuda S, et al. Arteriosclerosis and Thrombosis 1992;12(4):494-502). VSMC also secrete into the extracellular matrixproteoglycans, collagen, and elastic fibers. Upon entry, monocytestransform into macrophages, take up lipids as multiple small inclusions,and become foam cells. Extracellular proteoglycans bind lipids andprogressively increase their lipid-binding capacity by extension oftheir disaccharide arms. Some factors promote the death of macrophagesand VSMC. The necrotic debris provokes further inflammation. Increasingaccumulation of extracellular lipids coalesces into pools and causescell necrosis. Fibrotic tissue forms a fibrous cap over the lipid-richnecrotic core. (See id.). New vaso vasorum with thin walls invade thediseased intima from the media. These fragile vessels of endothelium,lacking pericytes for support, may leak, producing hemorrhage within thearterial wall. These intramural hemorrhages provoke increased fibroustissue deposition. Calcium deposits in the wall occur throughout allthese steps, initially as small aggregates, and later as large nodules.(See Insull W. American Journal of Medicine 2009; 122(1):S3-S14). It isalso known that type I, III, IV, V, and VI collagen are the maincollagens within atherosclerotic plaques but the distribution varies bystage and progression of the lesion. (See Katsuda S, et al.Arteriosclerosis and Thrombosis 1992; 12(4):494-502). Thus, it isapparent that formation of atherosclerotic plaques follows a complexcontinuum of events.

Percutaneous vascular interventions and plaque debulking technology totreat the manifestations of cardiovascular disease secondary toatherosclerosis do exist. Currently available percutaneous therapies forsevere atherosclerosis include angioplasty with or without stenting,cryoplasty, laser atherectomy, or remote atherectomy. The later categoryincludes the use of the Silverhawk™ or Rockhawk Atherectomy™ devices,the Rotablator™, the Pathway Jetstream™, and the Diamondback OrbitalAtherectomy™ system. Each of these therapies enlarges the lumen of theartery, thereby treating the underlying stenotic lesion. However, eachof these therapies induces some form of trauma to the vascular wall.Angioplasty and stenting restores lumen patency by forcing the plaqueagainst the wall of the artery under high pressure balloon inflations,thereby inducing significant trauma to the vascular wall. Cryoplastyreduces plaque burden by initiating apoptosis of the cells in theatherosclerotic plaque by freezing these cells to a temperature of −10°C. Laser atherectomy and mechanical remote atherectomy devices debulkthe plaque but do so in association with high thermal temperatures. Infact, the Rotablator rotational atherectomy device was found to resultin temperature increases of 2-4° C. with minimal decelerations, butincreases of 11-14° C. with continuous ablation or rapid decelerations.Therefore, each of the current FDA-approved therapies induces some formof mechanical injury to the vessel wall, which ultimately stimulates thedevelopment of neointimal hyperplasia and results in significantarterial restenosis. Furthermore, these therapies are costly, andrequire considerable time to debulk long segments of plaque. Therefore,new methodologies to reduce atherosclerotic plaques without inducingmechanical trauma to the arterial wall are desirable.

Here a new methodology for treating atherosclerotic lesions is proposedand developed through the optimization of a “digestion” solution thatwill result in non-traumatic in vivo digestion of atheroscleroticplaques. Given the fact that most atherosclerotic plaques are composedof lipids, proteoglycans, collagens, and calcium deposits, it ishypothesized that a “digestion” solution containing agents thatspecifically target these plaque components will dissolve and digest theplaque in vivo within a clinically relevant time frame, thereby allowingits use alone or in combination with other therapeutic interventions.Ultimately, the optimized digestion solution may be administered via adouble balloon occlusion catheter to an isolated segment in thevasculature percutaneously.

This proposed approach to treating severe atherosclerosis percutaneouslyis innovative, as no therapy exists on the market that is even remotelysimilar. Devices exist that debulk atherosclerotic plaques, as describedabove. However, each of these devices induces some form of thermal ormechanical trauma to the arterial wall. The presently disclosed plaquedigestion therapy is unique in that it will result in plaque debulkingwithout inducing any trauma to the vascular wall. Thus, this therapy,when successfully developed, has great potential to have a large impactin the clinical arena, given the prevalence of interventions foratherosclerosis.

SUMMARY

Disclosed are compositions and methods for treating a patient having orat risk for developing cardiovascular disease, including cardiovasculardisease secondary to atheroschlerosis. The disclosed compositions andmethods may include pharmaceutical compositions and therapeutic methodsfor treating atheroschlerotic plaques.

The disclosed compositions may include pharmaceutical compositionscomprising: (a) a mixture of collagenases comprising one, two, three,four, or more collagenases (e.g., collagenase type I, collagenase typeIII, collagenase type IV, and collagenase type V); and (b) a carrier.Preferably, the collagenases are present in the composition atconcentrations that are sufficient for digesting a human arterial plaqueand reducing mass of the human arterial plaque by at least 30%(preferably at least 40%, 50%, 60%, 70%, 80%, or 90%) after the sampleis contacted with the composition for no more than about 2 hours(preferably for no more than about 1 hour, or more preferably for nomore than 30, 20, 10, or 5 minutes). For example, preferably thecollagenases are present in the composition at concentrations that aresufficient for digesting a human arterial plaque and reducing mass ofthe human arterial plaque by at least 30% after the sample is contactedwith the composition for no more than 10 minutes.

The disclosed compositions may include a mixture of collagenasescomprising one, two, three, four, or more collagenases. In someembodiments, the compositions may comprise each of collagenase type I,collagenase type III, collagenase type IV, and collagenase type V at asuitable concentration (e.g., each at a concentration of at least about0.1 mg/ml (preferably at a concentration of at least about 0.2 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 20, 30, 40, or 50 mg/ml)).Suitable concentrations ranges may concentrations ranges within about0.1-50 mg/ml (e.g., 0.2-0.8 mg/ml). In some embodiments, thepharmaceutical composition may comprise additional collagenases. Inother embodiments, the pharmaceutical composition may comprise acollagenase mixture consisting of one or more of the followingcollagenases: collagenase type I, collagenase type III, collagenase typeIV, and collagenase type V, and the composition includes no othercollagenase.

The pharmaceutical compositions disclosed herein may include additionalingredients for digesting or dissolving atheroschlerotic plaques. Insome embodiments, the pharmaceutical compositions further comprise acyclodextrin (e.g., alpha-, beta-, or gamma-cyclodextrin and preferablybeta-cyclodextrin). The pharmaceutical composition may comprise thecyclodextrin at a suitable concentration. Suitable concentration rangesmay include, but are not limited to, a range of about 1-100 mM, andpreferably a range of about 5-25 mM.

The pharmaceutical compositions disclosed herein may comprise achelating agent for a divalent cation (e.g., EDTA). The pharmaceuticalcomposition may comprise the chelating agent at a suitableconcentration. Suitable concentration ranges may include, but are notlimited to, a range of about 0.5 mg/ml-2 mg/ml, and preferably a rangeof about 0.75-1.25 mg/ml.

The pharmaceutical compositions disclosed herein may comprise a divalentcation (e.g., Ca²⁺). The divalent cation may be added to the compositionas a salt. In some embodiments, the compositions comprise Ca²⁺ at aconcentration of about 1-25 mM.

The pharmaceutical compositions disclosed herein may comprisecholesterol esterase at a suitable concentration for hydrolyzingcholesterol esters, triacylglycerols, phospholipids, ceramides,lysophospholipids, or a mixture thereof in a human arterial plaque.Suitable concentration ranges may include, but are not limited to, arange of about 0.1-10 units/ml.

The pharmaceutical compositions disclosed herein may compriselipoprotein lipase at a suitable concentration for hydrolyzing lipidsinto free fatty acids and monoacylglycerol in a human arterial plaque.Suitable concentration ranges may include, but are not limited to, arange of about 30-10,000 units/ml.

The pharmaceutical compositions disclosed herein further may compriseapolipoprotein CII at a suitable concentration for functioning as aco-factor for lipoprotein lipase. Suitable concentration ranges mayinclude, but are not limited to, a range of about 1-20 units/ml.

The pharmaceutical compositions disclosed herein may comprise pepsin ata suitable concentration for digesting carbon bonds in proteins bycleaving preferentially after the N-terminal of aromatic amino acidssuch as phenylalanine, tryptophan, and tyrosine. Suitable concentrationranges may include, but are not limited to, a range of about 1-50 mg/ml.

The pharmaceutical compositions disclosed herein may comprisephospholipase A2 at a suitable concentration for hydrolyzing glycerol inhuman arterial plaque by releasing fatty acids from the second carbongroup of glycerol.

The pharmaceutical compositions disclosed herein may comprise tissueplasminogen activator (tPA) at a suitable concentration for convertingplasminogen to plasmin. Suitable concentration ranges may include, butare not limited to, a range of about 0.5-20 mg/ml (preferably a range ofabout 0.5-2 mg/ml, and more preferably a range of about 0.75-1.25mg/ml).

The pharmaceutical compositions disclosed herein preferably have asuitable pH for physiological conditions. For example, thepharmaceutical composition may have a pH within a range of about 7-8(and preferably within a range of about 7.2-7.6).

The disclosed compositions may be utilized in methods for treating apatient having or at risk for developing cardiovascular disease. Thedisclosed compositions may be utilized in methods for treating a patienthaving or at risk for developing atheroschlerosis or atheroschleroticplaques. In some embodiments, the disclosed methods include methods fordigesting a human arterial plaque in a patient, the methods comprisingcontacting the plaque with a pharmaceutical composition as contemplatedherein (e.g., a pharmaceutical composition comprising (a) a mixture ofcollagenases comprising one, two, three, four or more collagenases(e.g., collagenase type I, collagenase type III, collagenase type IV,and collagenase type V); and (b) a carrier). Preferably, thecollagenases are present in the composition at concentrations that aresufficient for digesting a human arterial plaque and reducing mass ofthe human arterial plaque by at least about 30% (preferably at least40%, 50%, 60%, 70%, 80%, or 90%) after the sample is contacted with thecomposition for no more than about 2 hours, 1 hour, 50 minutes, 40minutes, 30 minutes, 20 minutes, 10 minutes, or preferably 5 minutes. Insome embodiments of the methods, the pharmaceutical composition isadministered to the patient via a double-balloon occlusion catheterwhereby the composition contact an atheroschlerotic plaque in thepatient and dissolves or digests at least a portion of the plaque. Thecomposition then is aspirated from the site of the plaque together withany dissolved, digested, or dislodged plaque material. Optionally, thesite subsequently may be washed with a saline solution and furtheraspirated to remove any additional dissolved, digested, or dislodgedplaque material.

In some embodiments of the disclosed methods, the methods includeadministering a first solution to a patient and then subsequentlyadministering a second solution to the patient, before, concurrentlywith, or subsequently to administering the first solution. The firstsolution and the second solution may be the same or different. Forexample, the first solution may be a dissolution solution and the secondsolution may be a digestion solution, where the dissolution solution maycomprise one or more of the following agents, a cyclodextrin, achelating agent, calcium, and tPA, cholesterol esterase, lipoproteinlipase, apolioprotein CII, pepsin, phospholipase A2, or mixturesthereof, and the digestion solution comprises one or more of thefollowing collagenases: collagenase type I, collagenase type III,collagenase type IV, and collagenase type V. The first solution and thesecond solution typically are administered via a double balloonocclusion catheter whereby the first solution and the second solutioncontact an atheroschlerotic plaque in the patient. The first solutionand the second solution then are aspirated from the site of the plaquetogether with any dissolved, digested, or dislodged plaque material.Optionally, the site subsequently may be washed with a saline solutionand further aspirated to remove any additional dissolved, digested, ordislodged plaque material.

In some embodiments of the disclosed methods, the methods includeadministering ultrasonic energy to a patient, e.g., at the site of anarterial plaque. Ultrasonic energy may be administered at the site ofthe arterial plaque before, during, or after contacting the arterialplaque with the pharmaceutical compositions disclosed herein.Preferably, the ultrasonic energy facilitates dissolution of thearterial plaque. The ultrasonic energy may have a suitable frequency andmay be administered for a suitable period of time to facilitatedissolution of the plaque.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. illustrates digestion of atherosclerotic plaques after exposureto different concentrations of a mixture of type I, III, IV, and Vcollagenases at 37 degrees Celsius and pH 7.4. The optimal concentrationfor digestion at 2- and 4-hour time points is 2 mg/ml, but allconcentrations were able to fully digest the plaques by 24 hours.

FIG. 2. illustrates digestion of atherosclerotic plaques after exposureto different combinations of EDTA (1 mg/ml), (β-cyclodextrin (Cyclo, 10mMol), tPA (1 mg/ml) and Collagenase mixture type I, III, IV, V (Col, 2mg/ml) at 37 degrees Celsius, 225 rpm agitation, and ph 7.4. The firstcombination (#1) consisted of all four reagents together at once. Thesecond combination (#2) consisted of EDTA, {β-cyclodextrin, and tPAtogether, with removal of this solution at 1 hour, and then addition ofthe collagenase mixture. The third combination (#3) consisted of allfour agents together at once, followed by removal of the solution at 1hour, and then addition of the collagenase mixture. The most effectivecombination was #2 at 4 hours, though #3 was effective as well.

FIG. 3. illustrates digestion of plaque sections using varyingconcentrations of mixed collagenases solution.

FIG. 4. illustrates digestion of full plaques over time.

FIG. 5. illustrates digestion of plaque section using agitation.

FIG. 6. illustrates the effect of pH on digestion of plaque sections.

FIG. 7. illustrates the effect of temperature on digestion of plaquesections.

FIG. 8. illustrates digestion of plaque sections from differentpatients.

FIG. 9. illustrates the effect of EDTA on digestion of plaque sections.

FIG. 10. illustrates the effect of cyclodextrin on digestion of plaquesections.

FIG. 11. illustrates the effect of tPA on digestion of plaque sections.

FIG. 12. illustrates the effect of adding a “dissolution” solution(β-cyclodextrin, tPA, and EDTA) prior to adding a “digestion” solutioncomprising a collagenase mixture (I, III, IV, and V).

FIG. 13. illustrates the effect of adding a “dissolution” solution 5,10, or 15 minutes prior to adding a “digestion” solution.

FIG. 14. illustrates greater than 40% digestion of a humanatherosclerotic plaque within 60 min (and >60% digestion by 120 min) byoptimizing collagenase, EDTA, calcium, β-cyclodextrin, tPA, temperature,pH, agitation, and time.

FIG. 15. provides photographs of plaques before and after treatment withthe dissolution and digestion compositions after 30 minutes.

DETAILED DESCRIPTION

The subject matter disclosed herein is described using severaldefinitions, as set forth below and throughout the application.

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below, itis to be understood that as used in the specification, embodiments, andin the claims, “a”, “an”, and “the” can mean one or more, depending uponthe context in which it is used.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” or“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.”

As used herein, the term “patient” may be used interchangeable with theterm “subject” and means an animal, which may be a human or non-humananimal, in need of treatment. Non-human animals may include dogs, cats,horses, cows, pigs, sheep, and the like.

A “patient in need thereof” may include a patient having or at risk fordeveloping cardiovascular disease (e.g., cardiovascular diseasesecondary to atheroschlerosis). A patient in need thereof may refer to apatient having or at risk for acquiring an arterial disease or disordersuch as atherosclerosis or atheroschlerotic plaques. A patient in needthereof may refer to a patient having or at risk for acquiringneointimal hyperplasia or acute arterial thrombosis. A patient in needthereof may refer to a patient having recently undergone an angioplastyprocedure.

As used herein, the phrase “therapeutically effective amount” shall meanthat dosage of an active agent that provides the specificpharmacological response for which the active agent is administered in asignificant number of subjects in need of such treatment. Atherapeutically effective amount of an active agent that is administeredto a particular subject in a particular instance will not always beeffective in all instances for treating the conditions/diseasesdescribed herein, even though such dosage is deemed to be atherapeutically effective amount in particular instances by those ofskill in the art.

The compositions disclosed herein may be formulated as pharmaceuticalcompositions. For example, pharmaceutical compositions disclosed hereinmay include a carrier, excipient, or diluent (i.e., agents), which arenontoxic to the cell or mammal being exposed thereto at the dosages andconcentrations employed. Often a physiologically acceptable agent is anaqueous pH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The disclosed compositions may be administered to a patient via the useof a multiple balloon catheter as known in the art. For example,multiple balloon catheters are disclosed in U.S. Pat. No. 5,514,092, thecontent of which is incorporated herein by reference in its entirety.

In some embodiments of the methods disclosed herein, the methods includeadministering ultrasonic energy to a patient, e.g., administeringultrasonic energy at the site of an arterial plaque in the patient.Ultrasonic energy may be administered at the site of the arterial plaquebefore, during, or after contacting the arterial plaque with thepharmaceutical compositions disclosed herein. Preferably, the ultrasonicenergy has a suitable frequency (e.g., 1-1000 kHz, more preferably 5-500kHz, even more preferably 10-100 kHz) and may be administered for asuitable period of time to facilitate dissolution of the plaque (e.g,for 0-60, 0-50, 0-40, 0-30, 0-20, 0-10, or 0-5 minutes.).

Ultrasonic energy may be administered to the arterial plaque usingmethods known in the art. The use of ultrasound has also beendemonstrated to reduce atherosclerotic burden. Goyen et al. reported theuse of an intra-arterial ultrasound catheter to debulk atheroscleroticchronic total occlusions (“Intravascular ultrasound angioplasty inperipheral arterial occlusion. Preliminary experience.” Acta Radiol.2000;41(2)122-124, the content of which is incorporated herein byreference in its entirety.) Using fluoroscopic guidance, anover-the-wire catheter containing piezoelectric elements at its tip thatconvert electrical energy into ultrasound energy was slowly advancedthrough chronic total occlusion. The transmitter emitted continuousultrasound frequency of 42 kHz. During activation of the device, thecatheter tip was irrigated continuously with saline (8 ml/min) to removedebris. The authors reported 100% technical success in all 9 patients.The ultrasound catheter created a channel through the atheroscleroticplaque that was twice the diameter of the transmitter. The remainingplaque was treated with aspiration thrombectomy or balloon angioplasty.

Illustrative Embodiments

The following embodiments are illustrative and are not intended to limitthe scope of the claimed subject matter.

Embodiment 1

A pharmaceutical composition comprising: (a) a mixture of two, three,four or more collagenases (e.g., collagenase type I, collagenase typeIII, collagenase type IV, and collagenase type V); and (b) a carrier.

Embodiment 2

The pharmaceutical composition of embodiment 1, wherein the collagenasesare present in the composition at concentrations that are sufficient fordigesting a human arterial plaque and reducing mass of the humanarterial plaque by at least 30% (preferably at least 40%, 50%, 60%, 70%,80%, or 90%) after the sample is contacted with the composition for nomore than about 2 hours, 1 hour, 30, 20, 10, or 5 minutes.

Embodiment 3

The pharmaceutical composition of embodiment 1 or 2, comprising each ofcollagenase type I, collagenase type III, collagenase type IV, andcollagenase type V at a concentration of at least about 0.1 mg/ml(preferably at a concentration of at least about 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 20, 30, 40, or 50 mg/ml).

Embodiment 4

The pharmaceutical composition of any of embodiments 1-3, wherein themixture of collagenases consists of collagenase type I, collagenase typeIII, collagenase type IV, and collagenase type V.

Embodiment 5

The pharmaceutical composition of any of embodiments 1-4, furthercomprising a cyclodextrin (e.g., alpha-, beta-, or gamma-cyclodextrinand preferably beta-cyclodextrin).

Embodiment 6

The pharmaceutical composition of embodiment 5, wherein the cyclodextrinis present at a concentration of 1-100 mM (preferably 5-25 mM).

Embodiment 7

The pharmaceutical composition of any of embodiments 1-6, furthercomprising a chelating agent (e.g., EDTA).

Embodiment 8

The pharmaceutical composition of embodiment 7, wherein the chelatingagent is present at a concentration of 0.5 mg/ml-2 mg/ml (preferably0.75-1.25 mg/ml).

Embodiment 9

The pharmaceutical composition of any of embodiments 1-8, furthercomprising tissue plasminogen activator.

Embodiment 10

The pharmaceutical composition of embodiment 9, wherein the tissueplasminogen activator is present at a concentration of 0.5-20 mg/ml(preferably 0.75-1.25 mg/ml).

Embodiment 11

The pharmaceutical composition of any of embodiments 1-10, furthercomprising cholesterol esterase (preferably at a concentration of 0.1-10units/ml).

Embodiment 12

The pharmaceutical composition of any of embodiments 1-11, furthercomprising lipoprotein lipase (preferably at a concentration of30-10,000 units/ml).

Embodiment 13

The pharmaceutical composition of any of embodiments 1-12, furthercomprising apolipoprotein CII (preferably at a concentration of 1-20units/ml).

Embodiment 14

The pharmaceutical composition of any of embodiments 1-13, furthercomprising phospholipase A2.

Embodiment 15

The pharmaceutical composition of any of embodiments 1-14, furthercomprising pepsin (preferably at a concentration of 1-50 mg/ml).

Embodiment 16

The pharmaceutical composition of any of embodiments 1-15, wherein thecomposition has a pH of about 7-8 (preferably about 7.2-7.6).

Embodiment 17

A method for digesting a human arterial plaque in a patient, the methodcomprising contacting the plaque with a pharmaceutical compositioncomprising: (a) a mixture of collagenases comprising two, three, four ormore collagenases (e.g., collagenase type I, collagenase type III,collagenase type IV, and collagenase type V); and (b) a carrier.

Embodiment 18

The method of embodiment 17, wherein the collagenases are present in thecomposition at concentrations that are sufficient for digesting a humanarterial plaque and reducing weight of a sample of the human arterialplaque by at least 30% (preferably at least 40%, 50%, 60%, 70%, 80%, or90%) after the sample is contacted with the composition for no more thanabout 2 hours, 1 hour, 30, 20, 10, or 5 minutes.

Embodiment 19

The method of embodiment 17 or 18, comprising each of collagenase typeI, collagenase type III, collagenase type IV, and collagenase type V ata concentration of at least about 0.1 mg/ml (preferably at aconcentration of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1, 2, 4, 6, 8, 10, 20, 30, 40, or 50 mg/ml).

Embodiment 20

The method of any of embodiments 17-19, wherein the mixture ofcollagenases consists of collagenase type I, collagenase type III,collagenase type IV, and collagenase type V.

Embodiment 21

The method of any of embodiments 17-20, further comprising a (e.g.,alpha-, beta-, or gamma-cyclodextrin and preferably beta-cyclodextrin).

Embodiment 22

The method of embodiment 21, wherein the cyclodextrin is present at aconcentration of 1-100 mM (preferably 5-25 mM).

Embodiment 23

The method of any of embodiments 17-22, further comprising a chelatingagent (e.g., EDTA).

Embodiment 24

The method of embodiment 23, wherein the chelating agent is present at aconcentration of 0.5 mg/ml-2 mg/ml (preferably 0.75-1.25 mg/ml).

Embodiment 25

The method of any of embodiments 17-24, further comprising tissueplasminogen activator.

Embodiment 26

The method of embodiment 25, wherein the tissue plasminogen activator ispresent at a concentration of 0.5-20 mg/ml (preferably 0.75-1.25 mg/ml).

Embodiment 27

The method of any of embodiments 17-26, further comprising cholesterolesterase (preferably at a concentration of 0.1-10 units/ml).

Embodiment 28

The method of any of embodiments 17-27, further comprising lipoproteinlipase (preferably at a concentration of 30-10,000 units/ml).

Embodiment 29

The method of any of embodiments 17-28, further comprisingapolipoprotein CII (preferably at a concentration of 1-20 units/ml).

Embodiment 30

The method of any of embodiments 17-29, further comprising phospholipaseA2.

Embodiment 31

The method of any of embodiments 17-30, further comprising pepsin(preferably at a concentration of 1-50 mg/ml).

Embodiment 32

The method of any of embodiments 17-31, wherein the composition has a pHof about 7-8 (preferably about 7.2-7.6).

Embodiment 33

The method of any of embodiments 17-32, wherein the composition isadministered to the patient via a double-balloon catheter.

Embodiment 34

The method of any of embodiments 17-33, wherein the composition iscontacted with the human arterial plaque for at least 1 hour (or atleast 2 hours or 4 hours).

Embodiment 35

The method of any of embodiments 17-34, further comprising applyingultrasonic energy to the plaque.

Embodiment 36

A method of treating an atherosclerotic plaque in a patient, the methodcomprising administering a collagenase composition comprising two,three, four or more collagenases to the patient at the site of theatherosclerotic plaque via a double balloon occlusion catheter.

Embodiment 37

The method of embodiment 36, wherein the collagenase compositioncomprises a combination of collagenase types I, III, IV, and V.

Embodiment 38

The method of embodiment 36, wherein the collagenase compositioncomprises 0.5-50 mg/ml of each of collagenase types I, III, IV, and V.

Embodiment 39

The method of embodiment 36, wherein the collagenase compositioncomprises 1.0-10 mg/ml of each of collagenase types I, III, IV, and V.

Embodiment 40

The method of any of embodiments claim 36-39, further comprisingadministering a composition comprising tissue plasminogen activator(tPa), before, during, or after administering the collagenasecomposition.

Embodiment 41

The method of any of embodiments 36-40, further comprising administeringa composition comprising a cyclodextrin, before, during, or afteradministering the collagenase composition.

Embodiment 42

The method of embodiment 41, wherein the cyclodextrin isbeta-cyclodextrin.

Embodiment 43

The method of any of embodiments 36-42, further comprising administeringa composition comprising a chelator, before, during, or afteradministering the collagenase composition.

Embodiment 44

The method of any of embodiments 36-43, further comprising administeringa composition comprising tissue plasminogen activator (tPa), before,during, or after administering the collagenase composition.

Embodiment 45

The method of any of embodiments 36-44, further comprising administeringa composition comprising cholesterol esterase, before, during, or afteradministering the collagenase composition.

Embodiment 46

The method of any of embodiments 36-45, further comprising administeringa composition comprising lipoprotein lipase, before, during, or afteradministering the collagenase composition.

Embodiment 47

The method of any of embodiments 36-46, further comprising administeringa composition comprising apolipoprotein CII, before, during, or afteradministering the collagenase composition.

Embodiment 48

The method of any of embodiments 36-47, further comprising administeringa composition comprising phospholipase A2, before, during, or afteradministering the collagenase composition.

Embodiment 49

The method of any of embodiments 36-48, further comprising administeringa composition comprising comprising pepsin, before, during, or afteradministering the collagenase composition.

Embodiment 50

The method of any of embodiments 36-49, further comprising applyingultrasonic energy to the plaque.

EXAMPLES

The following examples are illustrative and are not intended to limitthe scope of the disclosed and claimed subject matter.

Example 1 Digestion and Dissolution Solutions for Arterial PlaquesBackground

The present disclosure outlines a new and unique method of removingatherosclerotic plaque from arteries. Cardiovascular disease,predominately secondary to atherosclerosis, is the leading cause ofdeath in the United States, accounting for 60% of total mortality in2002. Percutaneous vascular procedures that treat severe atherosclerosisoften utilize balloon angioplasty, which is the expansion of a ballooninside the narrowed artery to increase the lumenal area, followed bydeployment of a rigid or non-compliant metal stent to prevent theelastic recoil that occurs following balloon angioplasty. While thistechnique is commonplace, it fails to remove or reduce theatherosclerotic plaque burden. The remaining plaque burden, along withthe stimulation of the arterial injury response by balloon angioplasty,result in significant restenosis rates, requiring further interventionsor surgery, or myocardial infarction, limb loss, or death. Whiledrug-eluting stents have demonstrated a significant reduction inrestenosis rates, its safety has come under question due to increasedrates of late stent thrombosis. Here, new methods for treatingatherosclerosis are proposed via optimizing in vivo “digestion” ofatherosclerotic plaques, thereby removing atherosclerotic plaque burden.It is hypothesized that by removing the plaque burden, and by avoidingthe use of balloon angioplasty and its associated activation of thearterial injury cascade, restenosis rates following this approach willbe dramatically decreased compared to all current interventions aimed totreat severe atherosclerosis.

Atherosclerotic plaques form over the course of several decades througha process of cholesterol deposition, inflammatory responses, lipidaccumulation, and extracellular matrix production. In the center of theatherosclerotic lesions, foam cells and extracellular lipids form a coreregion, which is surrounded by a fibrous cap of smooth muscle cells anda collagen rich matrix. The devastating consequences of atherosclerosisoccur when the fibrous cap ruptures, exposing the thrombogenic core tothe blood, initiating coagulation with subsequent thrombosis of thevessel. Studies have revealed that plaques are not uniform when examinedbetween patients. Stary et al. have classified plaques based on theirdifferent characteristics: (I) Lipid-laden macrophages present as onlyisolated groups; (II) Lipid-laden macrophages stratified as adjacentlayers; (III) Macrophages, extracellular accumulation of lipids; (IV)Lipid core without fibrous tissue or fissure formation; (V) Smoothmuscle cells, extracellular matrix (collagen, glycoproteins,proteoglycans); (Va) Fibrous tissue, lipids; (Vb) Prominent calcificcomponent; (Vc) Prominent fibrous component; and (VI) Hemorrhage,fissure. (See Stary H C, et al., Circulation 1995; 92(5):1355-1374.

Using collagen type-specific antibodies, it is also known that type I,III, IV, V, and VI are the main collagens within atheroscleroticplaques, with the amount of distribution varying on the stage andprogression of the lesion. Here, new methods for treatingatherosclerotic lesions are proposed through optimization of a“digestion” solution that would promote in vivo digestion ofatherosclerotic plaques. Given the fact that most rigid plaques areconsistently composed of lipids, proteoglycans, collagens, and calciumdeposits, it is hypothesized that a “digestion solution” containingagents that specifically target plaque components will be able todissolve and digest the plaque in vivo in an accelerated amount of time.Furthermore, since the intimal layer of the arterial wall is separatedfrom the medial layer by the internal elastic lamina, avoidance of theuse of elastases will limit the plaque digestion to just the intimallayer, thereby preserving the medial layer of the arterial wall andsafely limiting the reaction in vivo.

Currently, the mainstay of therapy for severe atherosclerosis consistsof percutaneous balloon angioplasty with or without stenting, surgicalbypass grafting, or surgical endarterectomy, the later of which removesthe plaque burden surgically. The number of procedures performedannually for percutaneous approaches for the treatment of severeatherosclerosis greatly out number that of open surgery. In the currentera of endovascular options, many different percutaneous approachesexist. However, balloon angioplasty with or without stenting remains themost common approach utilized. This approach is plagued by the fact thatthe atherosclerotic plaque is not removed, but mechanically compressedagainst the arterial wall. The high inflation pressures required toaccomplish this in turn stimulates the arterial injury response in thevascular wall, which results in the development of neointimalhyperplasia that ultimately re-occludes the vessel. Thus, this procedurewhich aims to increase lumen area actually results in its own failure.For a long time, researchers have been trying to develop methods todebulk atherosclerotic plaques. Many different devices have beenintroduced into the market. Today, only a few debulking devices existthat are FDA approved. These include the Rotoblader™, the Silverhawk1™,the Rockhawk™, the Diamondback™, and the Jetstream™ device. Each ofthese devices mechanically removes or debulks the plaque either throughthe use of an oscillating blade that pulverizes the plaque or the use ofa blade that shaves the plaque. Each of these devices has drawbacks thatlimits it use, and none have proven to be superior to conventionalballoon angioplasty and stenting in large, prospective, randomizedclinical trials.

The presently proposed methods aim to remove the plaque burden withminimal trauma to the arterial wall and minimal risk to the patient. Thepresent inventors are unaware of these proposed methods ever having beenperformed in patients. The proposed methods do not require the use ofballoons or mechanical blades for contacting the plaque, thus avoidingthe potential risk of inducing arterial injury and at the same timeproviding a safe approach. All particulate matter will be aspirated,thereby avoiding embolization of atherosclerotic plaque, a problem thatall of the above-mentioned atherectomy devices face. Thus, when ourapproach is utilized, we expect superior outcomes that are associatedwith significantly reduce failure rates compared to currently availablemodalities, thereby diminishing the need for reintervention, improvingpatient outcomes, and reducing overall health care costs.

Materials, Methods, and Results

Atherosclerotic plaques from surgical endarterectomies were obtainedfrom various anatomic sites. All experiments were performed by cutting agiven plaque into equal parts by weight then exposing them to variousagents alone and in combination to determine the most optimal digestionsolution. The optimal concentration and conditions for plaquedissolution and digestion were determined for ethylenediaminetetraaceticacid (EDTA), β-cyclodextrin, tPA, collagenase type I, III, IV, and V.Weights of each specimen were obtained prior to exposure of eachsolution and at different time points to determine percent of digestedversus undigested plaque. Additional variables that were assessed andoptimized include: 1) different combinations of EDTA, β-cyclodextrin,and the collagenases, 2) temperature, 3) pH, and 4) agitation.

Initially, a digestion solution comprising type I, III, IV, and Vcollagenase at a concentration of 2 mg/ml was determined to be optimal(68% reduction, p<0.05) (FIG. 1). Next, different parameters of the“digestion solution” were tested to determine if the efficiency ofplaque digestion could be improved. Optimal conditions for the digestionsolution were found to be a temperature of 37 degree Celsius (51%reduction, p<0.05), pH 7.4 (47% reduction, p<0.05), and agitation (61%reduction, p<0.05). Using these standardized conditions for digestion,plaque-to-plaque variability was assessed with no significant differencein amount of digestion between different plaque specimens.

Next, agents that reacted with different components of the plaque weretested to further optimize digestion. The effect of localized chelationof calcium from the plaque was evaluated with exposure to EDTA. Theeffect of localized solubilization of extracellular lipid from theplaque was evaluated with exposure to β-cyclodextrin. And lastly,proteoglycans were dissolved by tissue plasminogen activator (tPA).Neither EDTA (0.5 mg/ml-2 mg/ml), β-cyclodextrin (1-100 mMol), or tPA(0.5-2 mg/ml) alone caused significant reduction in plaque weight (15%,2%, 20%, respectively, p<0.05). But when combined with collagenase indifferent combination schemes at optimal standardized conditions, thepercent digestion of plaque samples were improved (87% reduction at 4hrs, p<0.05) (FIG. 2).

Ideally, the dissolution and digestion solutions and methods ofadministration will be further improved to reduce the time for digestionin vivo. However, the concept of using a dissolution and digestionsolution to degrade an atherosclerotic plaque in vivo is sound andeffective. It is expected that the presently disclosed solution will beused to treat patients with severe atherosclerosis percutaneously viadelivery through the use of a double balloon occlusion catheter. Thesolution will be delivered after balloon occlusion is achieved. Thespace between the balloons will be irrigated with normal saline, thedigestion solution will be injected for a predetermined period of time,the digestion solution along with plaque debris will be aspirated, andflow will be restored. The methods will reduce the atheroscleroticplaque burden with no associated trauma to the vascular wall. Suitableadministration sites may include the internal elastic lamina, where,preferably, the disclosed compositions do not include elastases. Alldebris will be aspirated, thereby avoiding any risk of embolization.

Example 2 A New Paradigm for Treating Atherosclerotic Lesions

The following Example is derived from a abstract of a presentation givenat the 10^(th) Annual Arteriosclerosis, Thrombosis and Vascular BiologyConference on May 1, 2009.

Summary

Severe atherosclerotic disease is commonly treated via percutaneousinterventions, such as angioplasty, stenting, cryoplasty, or mechanicalatherectomy. However, these techniques either fail to reduceatherosclerotic plaque burden or reduce plaque burden without causingarterial injury. The aim of this study is to evaluate a novel method oftreating atherosclerotic plaques that does not induce injury to thevascular wall: in vitro plaque dissolution and digestion.

Methods

Atherosclerotic plaques were obtained from patients undergoingendarterectomies. The optimal concentration and conditions for plaquedissolution and digestion were determined for ethylenediaminetetraaceticacid (EDTA), β-cyclodextrin, and collagenase type I, III, IV, and V.Variables that were assessed included: 1) combination of differentagents, 2) time, 3) temperature, 4) pH, and 5) agitation. Weight of eachspecimen was measured at different time points to determine percent ofundigested plaque.

Results

It was determined that a combination of type I, III, IV, and Vcollagenase at a concentration of 4 mg/ml resulted in 48.2% reduction ofplaque at 2 hours (p<0.05). This combination was significantly moreeffective than each collagenase alone, or other combinations thereof(P<0.05). It also was determined that increasing temperature from 37° to44° Celsius increased efficacy by 10%, and that agitation (225 rpm)increased efficacy by 9% compared to stasis. Further, optimal pH (range7.2-7.7) was found to be 7.4 (47% reduction, p<0.05). Maximum plaquedigestion occurred upon first exposing the plaque to a dissolutionsolution consisting of β-cyclodextrin and EDTA for 15 minutes, thenchanging the solution to collagenases (I, III, IV, V) at a temperatureof 44° Celsius, pH 7.4, and agitation at 225 rpm (61% reduction, t=2hrs, p<0.05). Neither EDTA or β-cyclodextrin alone caused significantreduction in plaque weight (15% and 2%, respectively, p<0.05).

Conclusion

Effective plaque dissolution and digestion was observed in vitro. Thesedata serve as the foundation for further refinements of this techniquein order to reduce digestion time. This research has the potential todramatically alter standard approaches for percutaneous interventions totreat cardiovascular disease.

Example 3 Further Development of Digestion and Dissolution Solutions forArterial Plaques Methods, Results, and Discussion

A digestion solution that effectively results in 30-40% plaque digestionin vitro in 30 minutes was developed in a step-wise, methodical manner.First, collagenases were evaluated to determine feasibility of thedigestion solution. Human arterial plaque samples were collected andsectioned into approximately 1×1 cm sections. To determine percent ofplaque reduction, weights of the samples were measured at baseline andat designated time points. Types I, III, IV, and V collagenases werecombined (1:1:1:1) at concentrations of 0.5, 1, 2, and 4 mg/ml andtested against control solution (PBS). A collagenase solution at 4 mg/mlcaused the greatest percent reduction of plaque weight over controlafter 2 hrs in solution (50% versus 1%, respectively, p<0.05; FIG. 3).To determine if cutting the plaque into 1×1 cm sections enhanceddigestion compared to full plaques, digestion of full plaques wasevaluated. FIG. 4 shows that full plaques demonstrated similar reductionin plaque mass as compared to plaque sections (FIG. 4).

To establish optimal conditions for the collagenase solution, variousparameters were modified and evaluated with respect to enhancingdigestion. First, the effect of agitation on plaque reduction wasevaluated. Using the collagenase mixture, plaque specimens were exposedto static conditions or agitation with a shaker at 225 rpm. Plaquespecimens that were exposed to agitation had greater reduction versusstatic conditions (46% versus 37%, t=2 hrs; FIG. 5). The effect of pH onthe collagenase solution was then evaluated. Plaque specimens digestedwith a solution at pH 7.4 showed the greatest percent reduction (48%reduction at 2 hrs; FIG. 6). Finally, optimal temperature for thedigestion solution was established. Digestion was evaluated at roomtemperature, and between 37-40° C. The collagenase solution was moreeffective with increasing temperature (58% at 2 hrs; FIG. 7).

After implementing optimal digestion conditions with respect toagitation, pH, and temperature, the effect of the digestion solution wasevaluated on plaques harvested from different patients. Differences wereobserved wither respect to reduction in plaque mass when exposingdifferent plaque specimens to the digestion solution (FIG. 8),presumably due to intra-species plaque variability due to differentamounts of calcification, lipids, and plaque hemorrhage within theplaques from different patients. Thus various agents were tested incombination with collagenase to determine if an enhanced reduction ofplaque mass could be achieved regardless of intra-species variability.To evaluate the effect of localized chelation from the plaque,ethylenediaminetetraacetic acid (EDTA) was added to the collagenasesolution. Given that EDTA is a calcium chelator and that collagenaserequires calcium for activity, calcium chloride (5 mM) was added to thesolution. However, a collagenase solution having EDTA showed no enhancedreduction in plaque mass compared to a collagenase solution without EDTA(FIG. 9). To remove lipid components of the plaque, β-cyclodextrin(1-100 mM) was added to the collagenase solution. Plaque specimensdigested with collagenase and 10 mM β-cyclodextrin showed greatestreduction in plaque weight versus collagenase alone (39% versus 28% at 1hr; FIG. 10). The effect of tissue plasminogen activator (tPA) also wasassessed with respect to dissolving proteoglycans and thrombus withinthe plaque. Samples were exposed to collagenase, or collagenase with tPA(1-2 mg/ml). Tissue plasminogen activator at 2 mg/ml with collagenasedemonstrated the greatest percent reduction versus collagenase alone(37% vs. 27% at 1 hr; FIG. 11).

Given the potential of the various agents (i.e., EDTA, β-cyclodextrin,tPA, etc.) for enhancing plaque mass reduction, the agents wereevaluated further in various different combinations and differenttemporal arrangements with the collagenases. First, we tested whethercollagenase was more effective with all agents combined or if a“dissolution” solution consisting of these agents would enhance theeffect of collagenase if exposed to the plaque first. Indeed, the“dissolution” solution of EDTA, calcium, β-cyclodextrin, and tPA whenadded 1 hr prior to the “digestion” solution consisting of solely thecombination of the various collagenases was significantly more effectiveversus a combination of the “dissolution” and “digestion” solutiontogether (44% vs. 18% at 2 hrs; FIG. 12). To investigate how long tokeep the plaque in the “dissolution” solution prior to adding it to the“digestion” solution, it was observed that maintaining the plaque in the“dissolution” solution for 15 minutes then adding the “digestion”solution was optimal (FIG. 13). By optimizing collagenase, EDTA,calcium, β-cyclodextrin, tPA, temperature, pH, agitation, and time, wehave been able to achieve 40% digestion of a human atheroscleroticplaque within 60 min, and >60% digestion by 120 min (FIGS. 14 and 15).

Future Directions

Successful plaque digestion was performed in vitro with dissolution anddigestion solutions. Next, the dissolution and digestion solutions willbe evaluated on intact human atherosclerotic arteries using an ex vivoperfusion circuit with a goal of achieving significant plaque digestionin a clinically relevant time. Given that the presently disclosedmethods may be performed repeatedly, a 50% plaque digestion within 10minutes is clinically significant. To safeguard against the possibilityof digesting too much of the arterial wall, preferably the dissolutionand/or digestion solutions utilized in the methods do not includeelastases. Thus, digestion preferably is limited to the atheroscleroticplaque, above the internal elastic lamina.

An ex vivo perfusion circuit will be assembled from the followingcomponents: an LED micrometer, specialty chamber, computer, software,interface boards, Masterflow™ pump software, DB9 cable assemblies, L/SEasy Load™ pump head, L/S brushless programmable drive, MasterflexPharmed™ tubing LS/17, Masterflex™ silicone tubing LS/17, and Top Works™bottle top tubing. After successfully establishing the ex vivo perfusioncircuit, the efficacy of the dissolution and digestion solutions will beevaluated using intact human atherosclerotic arteries. Atheroscleroticarteries will be harvested from limb amputation specimens. After theartery is harvested, it will be weighed then secured to the perfusioncircuit. The artery will be preserved in a normal saline perfusate.Ultrasonography of the artery will be performed to quantify theatherosclerotic bulk. After documentation of the plaque, flow inside thelumen of the artery will be occluded with externally appliednon-traumatic vascular clamps. The digestion solution will be injectedinto the lumen of the artery via an angiocatheter through one end of theartery prior to occlusion. After 10-min increments, the digestionsolution will be evacuated and transferred to a 15 ml conical vial forlater evaluation. The artery will be weighed, returned to the perfusioncircuit, and undergo ultrasound assessment of the atheroscleroticplaque. Fresh digestion solution will be instilled and the process willbe repeated three times (i.e., 10-min, 20-min, and 30-min time points).The weight of the artery will be correlated with the atheroscleroticplaque bulk as measured by ultrasonography. After successfullyestablishing a baseline, the digestion solution will be optimized toachieve the greatest plaque reduction in the least amount of time. To dothis, the number of applications of fresh solution, time, temperature,pH, and agitation will be varied, similar to the in vitro studies. Inaddition, further refinements in the constituents and concentrations ofthe constituents of the dissolution and digestion solution will beperformed, as well as the inclusion of additional agents.

The studies described above are expected to: 1) demonstrate efficacy ofa digestion solution in an ex vivo perfusion circuit with a humanatherosclerotic artery, and 2) optimize the efficacy of digestionsolution to achieve 50% plaque digestion within 10 min in this ex vivoperfusion circuit. Additional agents that solubilize hydrophobicmolecules may be included in the dissolution or digestion solutions,such as cholesterol, or hydrolyzed cholesterol esters. Additionally,different proteases other than collagenases may be included.

In the foregoing description, certain terms have been used for brevity,clearness, and understanding. No unnecessary limitations are to beimplied therefrom beyond the requirement of the prior art because suchterms are used for descriptive purposes and are intended to be broadlyconstrued. The different compositions and method steps described hereinmay be used alone or in combination with other compositions and methodsteps. It is to be expected that various equivalents, alternatives andmodifications are possible. The cited patent and non-patent referencesare incorporated by reference in their entireties. The definitionsprovided in the present specification supersede any definition for aterm provided in a cited reference.

1. A pharmaceutical composition comprising: (a) a mixture comprisingeach of collagenase type I, collagenase type III, collagenase type IV,and collagenase type V; and (b) a carrier.
 2. The pharmaceuticalcomposition of claim 1, wherein the collagenases are present in thecomposition at concentrations that are sufficient for digesting a humanarterial plaque and reducing mass of the human arterial plaque by atleast 30% after the sample is contacted with the composition for no morethan about 30 minutes.
 3. The pharmaceutical composition of claim 1,comprising each of collagenase type I, collagenase type III, collagenasetype IV, and collagenase type V at a concentration of at least 0.5mg/ml.
 4. The pharmaceutical composition of claim 1, wherein the mixtureof consists of collagenase type I, collagenase type III, collagenasetype IV, and collagenase type V.
 5. The pharmaceutical composition ofclaim 1, further comprising a cyclodextrin at a concentration of 5-25mM.
 6. The pharmaceutical composition of claim 1, further comprising achelating agent at a concentration of 0.75-1.25 mg/ml.
 7. Thepharmaceutical composition of claim 1, further comprising tissueplasminogen activator at a concentration of 0.75-1.25 mg/ml.
 8. Thepharmaceutical composition of claim 1, further comprising cholesterolesterase at a concentration of 0.1-10 units/ml.
 9. The pharmaceuticalcomposition of claim 1, further comprising lipoprotein lipase at aconcentration of 30-10,000 units/ml.
 10. The pharmaceutical compositionof claim 1, further comprising apolipoprotein CII at a concentration of1-20 units/ml.
 11. The pharmaceutical composition of claim 1, furthercomprising phospholipase A2.
 12. The pharmaceutical composition of claim1, further comprising pepsin at a concentration of 1-50 mg/ml.
 13. Thepharmaceutical composition of claim 1, wherein the composition has a pHof about 7.2-7.6.
 14. A method for digesting a human arterial plaque ina patient, the method comprising contacting the plaque with thepharmaceutical composition of claim
 1. 15. The method of claim 14,wherein the composition is administered to the patient via adouble-balloon catheter
 16. The method of claim 14, wherein thecomposition is contacted with the human arterial plaque for no more thanabout 10 minutes.
 17. The method of claim 14, further comprisingapplying ultrasonic energy to the plaque.